culture media

Culture Media — History

Culturing methods were developed in the 19th century largely by Robert Koch and colleagues; animal/plant tissues were principal nutrient sources. Fanny Hesse's key contribution was showing that agar could form solidified culture media.

Components of a Culture Medium

1. Agars; 2. Peptones; 3. Meat and Plant Extracts; 4. Growth Factors; 5–6. Selective Components; 7–8. Differential Components & pH Buffers

Agars

Most common solidifying agent; a polysaccharide extract from marine algae. Melts at 84°C and solidifies at 38°C. 15.0 g/L = solid media; 7.5–10.0 g/L = soft/semisolid agars.

Peptones

Source of nitrogen; hydrolyzed proteins from meat scraps, beef muscle, gelatin, casein, and soybean meal.

Tryptone

Casein digest.

Phytone/Soytone

Soybean digest.

Peptone

Beef digest.

Peptone Concentration

0.1% concentration is often used as diluent.

Meat and Plant Extracts

Aqueous extracts containing amino acids, peptides, carbohydrates, vitamins, minerals, and trace metals.

Animal Extracts

Rich in protein and glycogen.

Plant Extracts

Rich in carbohydrates.

Brain Heart Infusion (BHI)

Example of a meat/plant extract medium; contains calf brains and beef heart.

Growth Factors

Enrich particular species; include vitamins, amino acids, fatty acids, trace metals, and blood components.

X Factor

Heme.

V Factor

NAD.

X Factor and V Factor

Can be supplied via hemoglobin, IsoVitaleX, and Supplement VX.

Selective Components

Inhibit nontarget microorganisms and are used to isolate specific organisms from mixed populations.

Toxic Chemicals Used as Selective Components

Bile salts, selenite, tetrathionate, tellurite, azide, phenylethanol, high NaCl, and dyes such as eosin, Crystal Violet, and Methylene Blue.

Antimicrobials Used as Selective Components

Ampicillin, chloramphenicol, colistin, cycloheximide, gentamicin, kanamycin, nalidixic acid, sulfadiazine, and vancomycin.

Differential Components

Differentiate microorganisms based on acid production from carbohydrates/carbon sources or amino acid decarboxylation.

pH Indicators and Chromogenic Dyes

Used to visualize metabolic reactions.

pH Buffers

Maintain the culture medium within a suitable pH range.

Types of Culture Media

1. Basal/Basic nutritive; 2. Enriched; 3. Enrichment; 4. Differential; 5. Selective; 6. Maintenance; 7. Reduced/Anaerobic; 8. Indicator media; 9. Selective agents.

1. Basal/Basic Nutritive Media

Supports growth of ordinary bacteria. Examples: peptone water, nutrient broth, nutrient agar.

2. Enriched Media

Basal medium + blood, serum, or egg yolk for fastidious bacteria. Examples: blood agar, serum agar, chocolate agar.

3. Enrichment Media

Liquid media that increase selective organisms that are outnumbered by unwanted species. Examples: selenite broth, tetrathionate broth, brilliant green broth.

4. Differential Media

Allows differentiation of various bacteria. Examples: MacConkey agar, EMB.

5. Selective Media

Contains inhibitory substances that suppress unwanted organisms. Examples: Deoxycholate Citrate Agar, Bismuth Sulfite Agar, Salmonella-Shigella Agar.

6. Maintenance Media

Maintains viability and physiological characteristics of a culture over time.

7. Reduced/Anaerobic Media

Contains reducing agents such as cysteine, thioglycollate, Na₂S, and sodium ascorbate that lower O₂/redox potential. Examples: cooked meat and thioglycollate medium.

8. Indicator Media

Contains fermentable sugars and pH indicators for presumptive identification. Examples: Litmus milk, TSI, BGA.

9. Selective Agents

Crystal violet, bile salts, brilliant green, potassium tellurite, sodium azide, and antibiotics.

Selective vs Differential Media

Selective media suppress unwanted growth; differential media help tell organisms apart, usually by color or reaction. A medium can be both selective and differential, such as MacConkey, Hektoen, and XLD.

Examples of Media Used in Diagnostic Bacteriology

1. Nutrient agar; 2. Blood agar; 3. MacConkey agar; 4. Brilliant green agar; 5. XLD agar; 6. TSI agar; 7. Selenite broth; 8. EMB agar; 9. Chocolate agar; 10. Edward's medium; 11. Hektoen enteric agar; 12. Simmons Citrate Agar; 13. Deoxycholate Citrate Agar; 14. Brain Heart Infusion (BHI); 15. Cooked Meat Medium; 16. Mueller-Hinton Agar (MHA); 17. Tetrathionate broth; 18. Thioglycollate broth; 19. Castañeda medium; 20. Smith-Baskerville medium; 21. Bordet-Gengou agar; 22. Lowenstein-Jensen (L-J).

Nutrient Agar

Basic medium for non-fastidious bacteria; used for colonial morphology, pigment production, and viable counting, e.g. Salmonella.

Blood Agar

Enriched medium for most bacteria including fastidious bacteria; sheep blood is added; detects hemolysis, e.g. Streptococcus, seen as a clear area on agar.

MacConkey Agar

For Enterobacteriaceae from the gut; used for water, food, and cheese testing for Salmonella and Shigella. Bile salts inhibit Gram(+) bacteria. pH indicator: neutral red. Substrate: lactose.

Brilliant Green Agar

Used for Salmonella isolation from feces; suppresses Gram(+) bacteria. Fermenters are yellow/green; S. typhi is reddish-white. Substrates: lactose and sucrose. Indicator: phenol red. Inhibitor: brilliant green dye.

XLD Agar

Isolates enteric pathogens such as Shigella, which forms red colonies with black centers. Sodium deoxycholate suppresses Gram(−) bacteria.

TSI Agar

Differentiates Gram(−) enterics by lactose, sucrose, and glucose fermentation.

Selenite Broth

Enrichment medium for Salmonella from feces, urine, water, and food. Sodium selenite inhibits Gram(+) and most Gram(−) bacteria including Enterococci; toxic at neutral pH.

EMB Agar

Selective/differential medium for fecal coliforms. E. coli produces a greenish metallic sheen; weak fermenters produce mucoid pink colonies.

Chocolate Agar

Non-selective enriched medium for Haemophilus, Neisseria, and Taylorella equigenitalis. Blood agar is heated, lysing RBCs and producing a brown color. Contains X-factor (Hemin) and V-factor (NAD).

Edward's Medium

Selective medium for Streptococci; involves aesculin hydrolysis and hemolysis. Substrates: aesculin and RBC. Inhibitors: crystal violet and thallous sulphate.

Hektoen Enteric Agar

Selective/differential medium for enteric pathogens. Bile salts and dyes inhibit Gram(+) bacteria; selective for Salmonella/Shigella from poultry, eggs, and dairy.

Simmons Citrate Agar

Used for differentiation among Enterobacteriaceae.

Deoxycholate Citrate Agar

Used for isolation of dysentery bacilli, food-poisoning Salmonella, and S. paratyphi B; superior to MCA for S. typhi. Heat-sensitive, so do not autoclave or remelt.

Brain Heart Infusion (BHI)

Nutrient-rich medium that cultivates fastidious organisms in blood culture and prepares inoculum for antimicrobial susceptibility testing (AST).

Cooked Meat Medium

Developed by Robertson for aerobes and anaerobes, including pathogenic clostridia.

Mueller-Hinton Agar (MHA)

Used for antimicrobial susceptibility testing; contains beef extract and casein acid hydrolysate.

Tetrathionate Broth

Selective enrichment medium for Salmonella.

Thioglycollate Broth

Multipurpose medium used to determine O₂ requirements; sodium thioglycolate depletes dissolved O₂.

Castañeda Medium

Biphasic medium for Brucella spp.

Smith-Baskerville Medium

Selective for B. avium and B. bronchiseptica; bromothymol blue indicator is green at pH 6.8.

Bordet-Gengou Agar

Blood-based medium for isolating B. pertussis; contains peptone, potato infusion, glycerol, and blood. Blood detects hemolysis; NaCl maintains osmotic equilibrium.

Lowenstein-Jensen (L-J)

Specific medium for Mycobacterium spp.

Media Suitable for Growth & Isolation of Specific Microorganisms

Actinomyces bovis → Brain Heart Infusion Glucose broth/agar, Thioglycollate broth (anaerobic); Bacillus anthracis → Almost all media; Bacteroides sp. → Blood agar, Thioglycollate broth (anaerobic); Blastomyces dermatitidis → Blood agar, Beef Infusion Glucose agar, Sabouraud's Glucose agar (+ antibiotic); Bordetella pertussis → Glycerin Potato, Bordet-Gengou agar; Brucella sp. → Trypticase Soy agar, Castañeda medium; Candida albicans → Sabouraud's Glucose agar; Clostridium sp. → Thioglycollate broth, Anaerobic Blood agar, Cooked Meat medium; Coccidioides immitis → Sabouraud's Glucose agar; Corynebacterium diphtheriae → Loeffler Blood serum medium, Cystine Tellurite Blood agar; Cryptococcus neoformans → Blood agar, Beef Infusion Glucose agar; E. coli and other coliforms → EMB, Endo agar, MacConkey; Francisella tularensis → Cystine Glucose Blood agar; Fungi/dermatophytes → Sabouraud's Dextrose agar, Corn Meal agar; Haemophilus influenzae → Chocolate agar; Histoplasma capsulatum → BHI Glucose broth, Sabouraud's Glucose agar; Mycobacterium tuberculosis → Lowenstein Jensen Medium, Petrogani medium; Nocardia asteroides → Beef Infusion Blood agar; Proteus vulgaris → EMB, MacConkey; Pseudomonas aeruginosa → Blood agar, EMB, MacConkey; Rickettsia and Chlamydia → Chick embryo, tissue culture; Salmonella, Shigella → Deoxycholate agar, Salmonella-Shigella agar, MacConkey, Tetrathionate broth.